Barbituric acid derivative and preventive and therapeutic agent for bone and cartilage containing the same

ABSTRACT

A compound of the formula                    
     wherein the substituents are as defined in the specification and solvates and salts thereof useful for treating bone and cartilage diseases.

This application is a 371 of PCT/IB/00961, filed Mar. 23, 1999.

The present invention relates to a novel barbituric acid derivative andthe method of the prevention and treatment by using the same ofmalignant hypercalcemia, osteolytic diseases such as Paget's disease ofbone and osteoporosis, such diseases with the denaturation and necrosisof a cartilage as osteoarthritis, necrosis of head of femur, andarticular rheumatism occurring in a knee, shoulder, and joint.

Japan has been gradually shifting to an aging society and, therefore,bone resorption diseases such as osteoporosis are becoming a big socialproblem. The bone resorption diseases are diseases of bone caused byabnormal sthenia of bone resorption exemplified by malignanthypercalcemia caused by myeloma and lymphoma, bone Paget's disease bylocal bone resorption, and osteoporosis caused by many factors such asaging and menopause. Although, at present, a female hormone, calcitonin,activated vitamin D3, parathyroid hormone, bisphosphonate, andipriflavone, etc. are being used for therapeutic treatment, these agentshave some defects respectively so as to remain as a symptomatic therapy.

On the other hand, osteoarthritis, necrosis of head of femur, and jointrheumatism are a disease occurring a defect of cartilage and bone causedby denaturation and necrosis of joint cartilage and subcartilaginousbone by various factors such as mechanical stresses, senescence,inflammation. The defect of bone largely affects the lowering of thequality of life through the deformation and pain of a joint. Any agentshave not substantially existed to inhibit or repair effectively thedefect of cartilage for the diseases.

Barbituric acid derivatives are a group of compounds which are used formedicinal drugs, agricultural chemicals, and many other purposes, andmany derivatives are known.

For example, there are an anticancer drug (refer to WO9116315),photosensitive materials for electronic photography (refer to JapanesePatent Application Laid-open Nos. 179361/91 and 111852/91), a vermifuge(refer to EP 192180), insecticides (EP 455300, DE 3903404, and GB1339748), herbicides (U.S. Pat. No. 4,797,147 and Japanese PatentApplication laid-open No. 154275/75), and a bactericide (EP 517660).

The present inventors first elucidated the structure and the stronginhibiting action of bone resorption, which had not been previouslyknown as far as the inventors' knowledge is concerned, of a series ofcompounds related to the present invention.

The inventors of the present invention formerly found substances showinga strong inhibiting action of bone resorption among derivatives ofpolyhydroxyphenol (Japanese Patent Application No. 137991/97). As aresult of further research and an analysis of structure-activityrelationship of those compounds, the inventors found a strong activityof a novel barbituric acid derivative and finally completed the presentinvention.

Thus, the purpose of the present invention is to provide a novel anduseful barbituric acid derivative, the salt thereof and the solvatethereof, and a method of prevention and treatment of various diseasesaffecting bones and cartilages by using these compounds.

The subject of the invention is a barbituric acid derivative representedby the following general formula (I), the salt thereof or the solvatethereof:

(wherein each of R or R¹ independently represents a hydrogen atom, asubstituted or an unsubstituted alkyl group or an alkenyl group ofC₁-C₁₅, a substituted or an unsubstituted arylmethyl group, or asubstituted or an unsubstituted aryl group, and R² represents asubstituted or an unsubstituted alkyl group or an alkenyl group ofC₁-C₁₅, a substituted or.

The present invention relates to a barbituric acid derivative shown insaid general formula (I), the salt thereof or the solvates thereofcontaining one or more compounds, and a composition of a medicinal drugcontaining a carrier that is acceptable for drug manufacturing,preferably, a composition of a medicinal drug being a preventive andtherapeutic agent for diseases affecting bones and cartilages.

Further, the present invention relates to a use of a barbituric acidderivative shown in said general formula (I), the salt thereof or thesolvate thereof containing one or more compounds for manufacturing acomposition of a medicinal drug for prevention and therapy of diseasesaffecting bones and cartilages.

Furthermore, the present invention relates to a method for preventionand therapy of diseases affecting bones and cartilages by administrationof an effective amount of a barbituric acid derivative shown in saidgeneral formula (I), the salt thereof or the solvate thereof containingone or more compounds for prevention and therapy of diseases affectingbones and cartilages.

In the compound shown in said general formula (I) of the presentinvention, alkyl groups are straight-chain or branched alkyl groups of1-15 carbon atoms, preferably 2-10, more preferably 4-10, and alkenylgroups are straight-chain or branched unsaturated hydrocarbon groups of2-15 carbon atoms, preferably 3-10, more preferably 4-10; preferably,unsaturated hydrocarbon groups of one or more carbon—carbon doublebonds.

In the compound shown in said general formula (I) of the presentinvention, aryl groups are monocyclic, polycyclic, or condensed ringaryl groups of 6-30 carbon atoms, preferably 6-20, more preferably 6-10;aryl groups of the present invention are monocyclic, polycyclic, orcondensed ring heterocyclic aromatic groups of at least one or morenitrogen atoms, oxygen atoms, or sulfur atoms in an aromatic ring, ofwhich size of one ring is 5-20 members, preferably 5-10 members, morepreferably 5-7 members, and may be condensed with a ring consisted ofsaturated or unsaturated hydrocarbon groups.

In the compound shown in said general formula (I) of the presentinvention, arylmethyl groups are exemplified by methyl groups made bysubstitution of said aryl group.

In said general formula (I) of the present invention, alkyl groups,alkenyl groups, arylmethyl groups, or aryl groups may be substituted bya substitution group that does not inhibit activities in medicalpurposes of the present invention.

The compound shown in said general formula (I) of the present inventionmight be protected by a protecting group allowing production of anactive body in a living body to express a physiological activity in theliving body.

Substitution groups of said alkyl groups, alkenyl groups, arylmethylgroups, or aryl groups in said general formula (I) of the presentinvention can be one made by substitution of these groups each other,when mutual substitution of these groups is possible. Examples are arylgroups substituted by an alkyl group, arylmethyl groups substituted byan alkyl group, alkyl groups substituted by an aryl group, and alkenylgroups substituted by an aryl group.

Other substitution groups are exemplified by a hydroxyl group, an aminogroup, an alkoxy group having said alkyl groups, an alkylthio group, amono- or a dialkylamino group, halogen atoms such as chlorine, bromine,and fluorine, alkylenedioxy groups such as a methylenedioxy group and a2,2-dimethylmethylenedioxy group, a cyano group, and a nitro group.

Preferable substitution groups are exemplified by lower alkyl groupssuch as a methyl group, an ethyl group, an n-propyl group, an isopropylgroup, an n-butyl group, and a t-butyl group, aryl groups such as aphenyl group and a naphthyl group, lower alkoxy groups such as a methoxygroup, an ethoxy group, and an n-propoxy group, di- lower alkylaminogroups such as a dimethylamino group, a diethylamino group, and adipropylamino group, halogen atoms such as iodine, bromine, chlorine,and fluorine, alkylenedioxy groups such as a methylenedioxy group, a2,2-dimethylmethylenedioxy group, a hydroxyl group, an amino group, acyano group, and a nitro group, etc.

Aryl groups in the compounds shown in said general formula (I) of thepresent invention are exemplified by a phenyl group, a naphthyl group,an anthracenyl group, a pyridyl group, a quinolyl group, a thienylgroup, and a pyrrolyl group.

Substitution groups of a substituted alkyl group or a substitutedalkenyl group are preferably specified by an atomic group introduced inreplacement to a hydrogen atom in preparing a derivative by substitutionof a hydrogen atom of these groups to another atom group. Thesubstitution groups can be a hydroxyl group, an alkoxy group, iodine,bromine, chlorine, and fluorine, an amino group, a cyano group, and anitro group. Substitution groups of a substituted arylmethyl group arepreferably atomic groups introduced in replacement to a hydrogen atom,when a derivative is prepared by substituting another atomic group of aresidue formed by removing a single hydrogen atom of the benzene ring ofan aromatic compound to a hydrogen atom of a compound of which hydrogenatom has been substituted by a methyl group. These substitution groupsare exemplified by a hydroxyl group, an alkoxy group, iodine, bromine,chlorine, fluorine, an amino group, a cyano group, and a nitro group.Substitution groups for substituting an aryl group are preferablyexemplified by those in which a residue group prepared by removing asingle hydrogen atom from the benzene ring of an aromatic compound hasbeen substituted by a hydroxyl group, an alkoxy group, iodine, bromine,chlorine, fluorine, an amino group, a cyano group, and a nitro group,etc.

Alkyl groups substituted or unsubstituted shown in said general formula(I) of the substitution groups R and R¹ of a barbituric acid derivativeare preferably exemplified by a methyl, an ethyl, a propyl, a butyl, apentyl, a hexyl, a heptyl, an octyl, a nonyl, a decanyl, an undecanyl, adodecyl, a tridecyl, a tetradecyl, or a pentadecyl group, etc.; inaddition to their isomers, those substituted or unsubstituted to thesegroups by said substitution group are preferable.

Alkenyl groups substituted or unsubstituted—shown in said generalformula (I) of the substitution groups R and R¹ of a barbituric acidderivative are preferably exemplified by an ethenyl, a propenyl, abutenyl, a pentenyl, a hexenyl, a heptentyl, an octenyl, a nonenyl, adecenyl, an undecenyl, a dodecenyl, a tridecenyl, a tetradecenyl, or apentadecenyl group, etc.; in addition to their isomers, thosesubstituted or unsubstituted to these groups by said substitution groupare preferable.

Arylmethyl groups substituted or unnsubstituted shown in said generalformula (I) of the substitution groups R and R¹ of a barbituric acidderivative are preferably exemplified by a phenylmethyl, anaphthylmethyl, an anthracenylmethyl, a pyridylmethyl, a quinolylmethyl,a thienylmethyl, or a pyrrolylmethyl group, etc.; in addition, thosesubstituted to these groups by said substitution groups and preferablyexemplified by those substituted or unsubstituted to these groups bysaid substitution groups such as a hydroxyl group, an alkoxy group,iodine, bromine, chlorine, fluorine, an amino group, a cyano group, or anitro group, etc.

Alkyl groups substituted or unsubstituted shown in said general formula(I) of the substitution groups R² of a barbituric acid derivative arepreferably exemplified by a methyl, an ethyl, a propyl, a butyl, apentyl, a hexyl, a heptyl, an octyl, a nonyl, a decanyl, an undecanyl, adodecyl, a tridecyl, a tetradecyl, or a pentadecyl group, etc.; inaddition to their isomers, those substituted or unsubstituted to thesegroups by said substitution group are preferable.

Alkenyl groups substituted or unsubstituted shown in said generalformula (I) of the substitution groups R² of a barbituric acidderivative are preferably exemplified by an ethenyl, a propenyl, abutenyl, a pentenyl, a hexenyl, a heptentyl, an octenyl, a nonenyl, adecenyl, an undecenyl, a dodecenyl, a tridecenyl, a tetradecenyl, or apentadecenyl group, etc.; in addition to their isomers, thosesubstituted or unsubstituted to these groups by said substitution groupare preferable.

Arylmethyl groups substituted or unsubstituted shown in said generalformula (I) of the substitution groups R² of a barbituric acidderivative are preferably exemplified by a phenylmethyl, anaphthylmethyl, an anthracenylmethyl, a pyridylmethyl, a quinolylmethyl,a thienylmethyl, or a pyrrolylmethyl group, etc.; in addition, thosesubstituted to these groups by said substitution groups and preferablyexemplified by those substituted or unsubstituted to these groups bysaid substitution groups such as a hydroxyl group, an alkoxy group,iodine, bromine, chlorine, fluorine, an amino group, a cyano group, or anitro group, etc.

Aryl groups substituted or unsubstituted shown in said general formula(I) of the substitution groups R² of a barbituric acid derivative arepreferably exemplified by a phenyl, a naphthyl, an anthracenyl, apyridyl, a quinolyl, a thienyl group, or a pyrrolyl group, etc.; inaddition, those substituted or unsubstituted to these groups by saidsubstitution groups are preferable.

Preferable compounds of the compound shown in said general formula (I)of the present invention are specifically exemplified as follows:

1,3-dibenzyl-5-(3-methyl-1-oxobutyl)barbituric acid (Compound No. 1),

1,3-dibenzyl-5-(phenylacetyl)barbituric acid (Compound No. 2),

1,3-dibenzyl-5-(2-thienylacetyl)barbituric acid (Compound No. 3),

1,3-bis(3-methylbutyl)-5-(3-methyl-1-oxobutyl)barbituric acid (CompoundNo. 4),

1-benzyl-3-(3-methylbutyl)-5-(3-methyl-1-oxobutyl)barbituric acid,

1-benzyl-3-(3-methylbutyl)-5-(phenylacetyl)barbituric acid,

1-benzyl-3-(3-methylbutyl)-5-(2-thienylacetyl)barbituric acid,

1,3-bis(3-methyl-2-butenyl)-5-(3-methyl-1-oxobutyl)barbituric acid,

1,3-bis(3-methyl-2-butenyl)-5-(phenylacetyl)barbituric acid,

1,3-bis(3-methyl-2-butenyl)-5-(2-thienylacetyl)barbituric acid,

1-benzyl-3-(3-methyl-2-butenyl)-5-(3-methyl-1-oxobutyl)barbituric acid,

1-benzyl-3-(3-methyl-2-butenyl)-5-(phenylacetyl)barbituric acid,

1-benzyl-3-(3-methyl-2-butenyl)-5-(2-thienylacetyl)barbituric acid,

1-(3-methyl-2-butenyl)-3-(3-methylbutyl)-5-(3-methyl-1-oxobutyl)barbituricacid,

1-(3-methyl-2-butenyl)-3-(3-methylbutyl)-5-(phenylacetyl) barbituricacid,

1-(3-methyl-2-butenyl)-3-(3-methylbutyl)-5-(2-thienylacetyl) barbituricacid.

The compound shown in said general formula (I) of the present inventioncan be synthesized by a conventional method for synthesis. For example,in a reaction formula represented by the following formula, synthesiscan be made easily by a condensation reaction of a barbituric acidderivative represented by the following formula (II) and an acylationagent represented by the following formula (III) (refer to the followingformula):

Acylation agents used are exemplified by acyl halides, acid anhydrides,or carboxylic acids, etc. It is well known by a person skilled in theart that condensation agents used for these reactions are individuallyselected according to a reaction applied. When an acylation agent (III)is acyl halides or acid anhydrides, a base lacking nucleophilicreaction, preferably a tertiary amine is used as a condensation agent.Examples are aromatic amines such as pyridine and quinoline, etc.,tertiary amines such as triethylamine, diisopropylethylamine, andN-methylpyrrolidine, etc., and aralkyl amines such as N,N-dimethylaniline, etc. A solvent is used for the reaction, however, it can beused that is not restrictive, but applicable for aniline, etc. A solventis used for the reaction; however, it can be used that is notrestrictive, but applicable for those inactive to both acylation agentand tertiary amine. Pyridine, and triethylamine, etc. are sometimes usedfor a condensation agent in combination of a solvent (refer to J. Org.Chem. 45: 4606, 1980; EP 455300; Japanese Patent Application laid-openNo. 102358/91; Japanese Patent Application laid-open No. 111852/91).

When an acylation agent (III) is a free carboxylic acid, oxyphosphoruschloride is used, for example, as a condensation agent (refer to GB1339748).

There are some methods for preparation of a barbituric acid derivative(II) to acylated in said reaction formula. Those methods are easilycontrived by a person skilled in the art. For example, the manufacturecan be conducted by the formula presented below (refer to the followingformula).

Examples are a method by condensation of an urea derivative (IV) and amalonic acid derivative (V) (Y and Z are halogen atoms such as chlorineor bromine, etc., or a hydroxyl group, or form an ester group togetherwith a carbonyl group), and a method by condensation of a carbodiimidederivative (VI) and malonic acid (V′) (refer to EP 455300).

The urea derivative (IV) and a carbodiimide derivative (VI), etc. arecompounds generally known as materials, and their preparation has beendescribed in the following reference in detail (Reference: S. R. Sandlerand W. Karo, Chapter 6/Ureas, Chapter 9/Carbodiimides, Academic Press,1986).

The bone resorption inhibiting activity of the barbituric acidderivative prepared according to an aforementioned method was tested bythe pit formation assay method. As a result, the compound of the presentinvention showed an excellent inhibiting ratio to bone resorption in theconcentration of 1×10⁻⁵ M.

Therefore, these compounds have a strong inhibiting activity to boneresorption. A barbituric acid derivative, the salt thereof, or thesolvates thereof shown by the general formula (I) of the presentinvention is useful as an effective ingredient of medicinal drug forprevention and therapeutic treatment of bone and cartilage diseases. Thepresent invention provides a composition of a medicinal drug containingone or more compounds as an effective ingredient, a method by using thecomposition for prevention and therapeutic treatment of various diseasesrelated to the inhibiting action of bone resorption, and a use of thesecompounds for preparation of the composition of a medicinal drug.

The present invention relates to a composition of a medicinal drugcontaining one or more compounds shown in said general formula (I), thesalt thereof or the solvate thereof, or a substance protected thereby(for example, a prodrug to become an active drug in a living body), thatare acceptable for drug manufacturing, as an effective ingredient. Thecomposition of a medicinal drug of the present invention relates to acomposition of a medicinal drug containing various carriers that isacceptable for drug manufacturing. The composition of a medicinal drugof the present invention has an activity of inhibiting bone resorptionand is useful as a preventive or therapeutic agent for diseasesaffecting bones and cartilages.

The diseases affecting bones and cartilages of the present inventioninclude bone resorption diseases such as malignant hypercalcemia, bonePaget's disease, and osteoporosis and diseases accompanying withdenaturation and necrosis of cartilage such as osteoarthritis, necrosisof head of femur, and joint rheumatism occurring in knee, shoulder, andhip joint.

A clinical administration dose as the effective ingredient of thepresent invention ranges in general 0.01 g-2 g a day (ca. 0.15 mg-30mg/kg/day) for an adult person as the compound of the present invention,preferably 0.1-2 g a day (ca. 1.5 mg-30 mg/kg/day) for an adult person,and more preferably 0.1-1 g a day (ca. 1.5 mg-15 mg/kg/day) for an adultperson, which are changeable according to a method of administration, astage of the disease, and a condition of a patient.

A possible administration method for the composition of a medicinal drugof the present invention includes intravenous, intramuscular, oral, andintrarectal administrations, in the intravenous administration, anintravenous drip can be applied in addition to a conventionalintravenous injection.

A drug manufacturing method for the composition of a medicinal drugcontaining the compound of the present invention is exemplified by aconventional method by using a conventional excipient and a conventionaladditive.

An injection preparation can be, for example, a powder preparation forinjection. In this case, a medicinal drug is prepared by adding one ormore appropriate water soluble excipients such as mannitol, sucrose,lactose, maltose, glucose, fructose, etc. to water to dissolve, dividinginto a vial or an ampoule followed by freeze drying and hermetic packingfor final preparation. In preparation for an oral administration, anenteric coated agent can be prepared in addition to a conventionaltablet, a capsule, a granule, a fine granule, and a powder.

In preparing the enteric coated agent, a tablet, a granule, or a finegranule can be prepared by adding, additives such as a lubricant such asmannitol, sucrose, lactose, maltose, starch, silica anhydride, calciumphosphate, etc., a binder such as carboxymethylcellulose,methylcellulose, gelatin, gum arabic, etc., and a disintegrant such ascalcium carboxymethylcellulose, followed by coating with one or moreenteric base agents such as cellulose, acetate phthalate,hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcelluloseacetyl succinate, polyvinyl alcohol phthalate, styrene, a copolymer ofmaleic acid anhydride, methyl-acrylate, and a copolymer ofmethylacrylate and, if necessary, adding a colorant such as titaniumoxide to make a preparation. A capsule preparation can be made byfilling enteric granules or fine granules prepared thereby in a capsule.On the other hand, it is possible that a capsule prepared by aconventional method is coated with said enteric base agent to makeenteric and made to an enteric capsule by using a capsule prepared byusing said enteric base agent singly or by mixing gelatin therewith.

For a suppository, it can be prepared by adding a semi-synthesized baseagent which is dissolved after preparation by mixing a monoglyceride offatty acid and a diglyceride of fatty acid in various proportions tocacao butter or triglyceride of fatty acid, followed by kneading mildlyand pour into a mold to form a suppository.

The present invention will now be described in detail with reference tothe following referencial examples, examples, and a test example that byno means limit the scope of the invention.

REFERENTIAL EXAMPLE 1 Preparation of N,N′-Dibenzylurea

Heated was a mixture of urea 5.84 g (9.72 mmol) and benzylamine 25.0 g(23.3 mmol, 2.4 equivalents) at 150° C.-155° C. for 5 hours attachingwith an air cooler. During the heating, observed was the ammonia gasevolution from colorless liquid reaction mixture. By cooling, it turnedsolidified at around room temperature. Purified was the reaction productby recrystallization from ethanol-benzene (1:1 mixture) to giveN,N′-dibenzylurea 14.0 g as white fine needles (59.9% yield).

NMR (DMSO-CDCl₃) δ4.33 (2H, d, J=5.8 Hz), 6.02 (1H, bt), 7.19-7.32 (5H,m).

REFERENTIAL EXAMPLE 2 Preparation of N,N′-Bis(3-methylbutyl)urea

Heated was a mixture of urea 0.601 g (10.0 mmol) and 3-methylbutylamine5.0 mL (ca. 6 equivalents) at 150° C. for 24 hours attaching with areflux cooler and during which time added more 3-methylbutylamine 2.5mL. Removed was the excess amine under the reduced pressure and allowedto cool to room temperature to give a colorless solid. Purification byrecrystallization from ethyl ether gave N,N′-bis(3-methylbutyl)urea 1.29g as white plates (64.5% yield).

NMR (CDCl₃) δ0.91 (6H, d, J=6.9 Hz), 1.38 (2H, q, J=7.2 Hz), 1.63 (1H,m), 3.15 (2H, t, J=7.3 Hz).

REFERENTIAL EXAMPLE 3 Preparation of 1,3-Dibenzylbarbituric Acid

Heated was a mixture of N,N′-dibenzylurea 12.02 g (50.0 mmol) andmalonic acid 5.20 g (50.0 mmol) in acetic anhydride 50.0 mL (530 mmol,10.6 equivalents) at 70° C.-75° C. for 14 hours under a nitrogenatmosphere. Then, removed were the volatile materials under the reducedpressure and distributed the residue between layers of 2 M sodiumhydroxide 250 mL and ether 200 mL. Separated was the alkaline aqueouslayer and after washing with ether, neutralized with concentratedhydrochloric acid under cooling in ice-water bath (pH 2-3). Extractedwas the neutrallized aqueous solution with chloroform, washed withwater, and dried over sodium sulfate. Removal of the solvent underreduced pressure gave 1,3-dibenzylbarbituric acid 14.7 g as pale yellowcrystalline powder (95.6% yield).

NMR (CDCl₃) δ3.60 (1H, s), 5.00 (2H, s), 7.22-7.45 (5H, m).

REFERENTIAL EXAMPLE 4 Preparation of 1,3-Bis(3-methylbutyl)barbituricAcid

Heated was a mixture of N,N′-bis(3-methylbutyl)urea 1.28 g (6.39 mmol)and malonic acid 665 mg (6.39 mmol) in acetic anhydride 6.4 mL (68 mmol,11 equivalents) at 70° C.-75° C. under a nitrogen atmosphere for 4hours. Removed were the volatile materials under the reduced pressureand distributed the residue between layers of 2 M sodium hydroxide 50 mLand ether 60 mL. Separated was the alkaline aqueous layer and afterwashing with ether, neutralized aqueous solution with concentratedhydrochloric acid (pH 2-3). Extracted was the neutrallized aqueoussolution with dichloromethane, washed with water, and dried over sodiumsulfate. Removal of the solvent under the reduced pressure gave orangesolid 1.22 g. Purified was the crude product by column chromatography(silica gel, ether elution) to obtain 1,3-bis(3-methylbutyl)barbituricacid 954 mg as pale yellow crystals (95.6% yield).

NMR (CDCl₃) δ0.95 (6H, d, J=6.6 Hz), 1.48 (2H, q, J=7.8 Hz), 1.62 (1H,m), 3.65 (1H, s), 3.87 (2H, t, J=7.8 Hz).

EXAMPLE 1 Synthesis of 1,3-Dibenzyl-5-(3-methyl-1-oxobutyl)barbituricAcid (Compound No. 1)

To a solution of 1,3-dibenzyl barbituric acid 265 mg (0.859 mmol) in drydichloromethane 2.0 mL under a nitrogen atmosphere, added was pyridine1.0 mL (12 mmol) and stirred at 0° C. Added was isovaleryl chloride 104mg (0.858 mmol, 1.00 equivalent) in dichloromethane 1.0 mL to thesolution slowly during a period of 20 minutes, then stirred at roomtemperature for 3 hours. Added was dichloromethane 50 mL and washed theorganic layer with 2 M hydrochloric acid and saturated brine, and driedover sodium sulfate. Purified was the crude product 351 mg, afterremoval of the solvent under the reduced pressure, by columnchromatography (silica gel, eluted with dichloromethane). It obtained1,3-dibenzyl-5-(3-methyl-1-oxobutyl)barbituric acid 299 mg as colorlessviscous oil at first, and in 2 days it turned white plates (88.7%yield).

NMR (CDCl₃) δ1.01 (6H, d, J=6.6 Hz), 2.20 (1H, m), 3.04 (2H, d, J=6.8Hz), 5.10 (2H, s), 5.11 (2H, s), 7.23-7.47 (10H, m).

EXAMPLE 2 Synthesis of 1,3-Dibenzyl-5-(phenylacetyl)barbituric Acid(Compound No. 2)

To a solution of 1,3-dibenzyl barbituric acid 192 mg (0.623 mmol) in drydichloromethane 2.0 mL under nitrogen atmosphere, added was pyridine 1.0mL (12 mmol) and stirred at 0° C. Added was phenylacetyl chloride 96 mg(0.623 mmol, 1.00 equivalent) in dichloromethane 1.0 mL to the solutionslowly during a period of 20 minutes, then stirred at room temperaturefor 3 hours. Added was dichloromethane 40 mL and washed the organiclayer with 2 M hydrochloric acid and saturated brine, and dried oversodium sulfate. Purified was the crude product 273 mg, after removal ofthe solvent under the reduced pressure, by column chromatography (silicagel, eluted with dichloromethane). It obtained1,3-dibenzyl-5-(phenylacetyl)barbituric acid 236 mg as colorless viscousoil at first (88.7% yield) and it gradually turned white plates.

NMR (CDCl₃) δ4.51 (2H, s), 5.09 (2H, s), 5.12 (2H, s), 7.25-7.60 (15H,m).

EXAMPLE 3 Synthesis of 1,3-Dibenzyl-5-(2-thienylacetyl)barbituric Acid(Compound No. 3)

To a solution of 1,3-dibenzylbarbituric acid 154 mg (0.500 mmol) in drydichloromethane 2.0 mL under a nitrogen atmosphere, added was pyridine1.0 mL (12 mmol) and stirred at 0° C. Added was 2-thienylacetyl chloride80 mg (0.50 mmol, 1.00 equivalent) in dichloromethane 1.0 mL to thesolution slowly during a period of 20 minutes, then stirred at roomtemperature for 3 hours. Added was ether 50 mL and washed the organiclayer with 2 M hydrochloric acid and saturated brine, and dried oversodium sulfate. Purified was the crude product 216 mg after removal ofthe solvent under the reduced pressure, by column chromatography (silicagel, eluted with dichloromethane). It obtained1,3-dibenzyl-5-(2-thienylacetyl)barbituric acid 153 mg as yellow viscousoil (56.9% yield).

NMR (CDCl₃) δ4.68 (2H, s), 5.09 (2H, s), 5.13 (2H, s), 6.94 (1H, dd,J=3.5, 5.3 Hz), 7.03 (1H, dd, J=1.1, 3.5 Hz), 7.20 (1H, dd, J=1.1, 5.3Hz), 7.23-7.50 (10H, m).

EXAMPLE 4 Synthesis of1,3-Bis(3-methylbutyl)-5-(3-methyl-1-oxobutyl)barbituric Acid (CompoundNo. 4)

To a solution of 1,3-bis(3-methylbutyl)barbituric acid 268 mg (1.00mmol) in dry dichloromethane 3.0 mL under a nitrogen atmosphere, addedwas pyridine 0.81 mL (0.79 g, 10 mmol, 10 equivalents) and stirred at 0°C. Added was isovaleryl chloride 121 mg (1.00 mmol, 1.00 equivalent) indichloromethane 1.5 mL to the solution slowly during a period of 20minutes, then stirred at room temperature for 4 hours. Added wasdichloromethane 40 mL and washed the organic layer with 2 M hydrochloricacid and saturated brine, and dried over sodium sulfate. Purified wasthe crude product 383 mg, after removal of the solvent under the reducedpressure, by column chromatography (silica gel, eluted withdichloromethane). It obtained1,3-bis(3-methylbutyl)-5-(3-methyl-1-oxobutyl)barbituric acid 278 mg aswhite plates (79.0% yield).

NMR (CDCl₃) δ0.97 (6H, d, J=6.6 Hz), 1.02 (12H, d, J=6.6 Hz), 1.52 (2H,m), 1.64 (2H, m), 2.20 (1H, m), 3.04 (2H, d, J=7.1 Hz), 3.91 (4H, m).

Test 1

(1) Preparation of cells

ICR mice 11-12 days old (Charles River) were subjected to euthanasiathrough ether anesthesia, and disinfected by dipping in 70% ethanolimmediately. Subsequently, a femur and a shank of a mouse was removed,chopped in an -MEM culture medium (Flow Labs Corp.) containing 5% FBS(Irving Scientific Corp.), 100 U/mL penicillin, and 100 g/mLstreptomycin. Supernatant obtained by pipetting was collected, washedwith culture medium solution, suspended in a solution containing 5% FBSand -MEM culture medium to yield osteocytes containing osteoclasts.Supernatant of osteocyte suspending solution was taken 3 minutes afterstanding still to pass through meshes (Cell Strainer, 70 m, FalconCorp.). The filtrate was adjusted to the concentration of 1 107 cells/mLto use for pit formation assay.

(2) Test by the pit formation assay

An ivory piece was cut into slices in 150 m thickness by using aprecision, low speed cutter (Buehler Corp.) and punched to make acylinderial holes in 6 mm diameter. The ivory piece was dipped in 70%ethanol, treated by sonication for 5 minutes twice, and washed withsterilized PBS three times and with culture medium twice. The ivorypiece was put in pits of a 96-pit culture plate (Falcon Corp.), 100 μlof culture medium, that contains a chemical compound of the presentinvention adjusted to a concentration of 2×10⁻⁵ M, was poured into eachpit, and 100 μL of culture medium, that contains 1×10⁷ osteocytes/mLpreviously prepared, into each pit (the final concentration of thecompound was 1×10⁻⁵ M), and finally cultivation was carried out in anincubator under 37° C. and 10% CO₂ for 3 days. Then cells on an ivorypiece were removed in 2 M sodium hydroxide solution, washed with waterand methanol to stain resorption cavity with Coomassie Brilliant Blueand count numbers of absorbed cavities under a microscope. Theinhibition rate of bone resorption was calculated on the basis thatresorption cavity was assumed 0% in the case of no addition of thecompound under the presence of rPTH (1×10⁻⁸ M) in the solution and thecase of no resorption cavity was assumed 100%. A good result was yieldedin the same experiment with variation of resorption cavity numbers amongexperimental groups due to a difference among proportions of variouscell in the solution, in which osteocytes were suspended, and also amongthe lot numbers of used animals.

Table 1 shows a result. As clearly shown by the result, the compound ofthe present invention shows a distinct inhibiting rate of boneresorption and is useful as a substance having an action to inhibit boneresorption.

TABLE 1 Number of resorption Number of cavity at no Added Concentrationresorption addition of Inhibiting Compound of Compound cavity Compoundrate No. (M) (mean ± SD) (mean ± SD) (%) 1 1 × 10⁻⁵ 1.0 ± 0.5 224.0 ±20.0 99.2 2 1 × 10⁻⁵ 2.2 ± 0.6 224.0 ± 20.0 99.0 3 1 × 10⁻⁵ 7.0 ± 1.7179.6 ± 34.4 96.7 4 1 × 10⁻⁵ 21.1 ± 4.3  224.0 ± 20.0 90.6

What is claimed is:
 1. A method of treating diseases of bones and cartilage in warm-blooded animals comprising administering to warm-blooded animals an amount of a compound selected from the group consisting of a compound of the formula

wherein R and R¹ are individually selected from the group consisting of unsubstituted or substituted alkyl and alkenyl of 3 to 15 carbon atoms and unsubstituted or substituted arylmethyl, R² is 2-thienylmethyl and unsubstituted or substituted arylmethyl, the substituents being at least one member of the group consisting of —OH, —NH₂, alkoxy of up to 10 carbon atoms, alkylthio of up to 10 carbon atoms, mono- and dialkylamino, halogen, —CN, —NO₂, alkylenedioxy, alkyl of up to 10 carbon atoms and aryl and its pharmaceutical salts sufficient to treat bone and cartilage disease.
 2. The method of claim 1 wherein R¹ is 2-thienylmethyl.
 3. The method of claim 1 wherein the active compound is selected from the group consisting of 1,3-dibenzyl-5-(3-methyl-1-oxobutyl)barbituric acid, 1,3-dibenzyl-5-(phenylacetyl)barbituric acid, 1,3-dibenzyl-5-(2-thienylacetyl)barbituric acid and 1,3-bis(3-methylbutyl)-5-(3-methyl-1-oxobutyl)barbituric acid.
 4. The method of claim 1 wherein the compound selected from the group consisting of 1,3-dibenzyl-5-(3-methyl-1-oxobutyl)barbituric acid, 1,3-dibenzyl-5-(phenylacetyl)barbituric acid, 1,3-dibenzyl-5-(3-thienylacetyl)barbituric acid, 1-benzyl-3-(3-methylbutyl)-5-(3-methyl-1-oxobutyl)barbituric-acid, 1-benzyl-3-(3-methylbutyl)-5-(phenylacetyl)barbituric acid, 1-benzyl-3-(3-methylbutyl)-5-(2-thienylacetyl)barbituric-acid, 1-benzyl-3-(3-methyl-2-butenyl)-5-(3-methyl-1-oxobutyl)barbituric acid, 1-benzyl-3-(3-methyl-2-butenyl)-5-(phenylacetyl)barbituric acid, and 1-benzyl-3-(3-methyl-2-butenyl)-5-(2-thienylacetyl)barbituric acid.
 5. A composition for treating bone and cartilage diseases comprising an amount of a compound selected from the group consisting of a compound of the formula

wherein R and R¹ are individually selected from the group consisting of unsubstituted or substituted alkyl and alkenyl of 3 to 15 carbon atoms and unsubstituted or substituted arylmethyl, R² is 2-thienylmethyl and unsubstituted or substituted arylmethyl, the substituents being at least one member of the group consisting of —OH, —NH₂, alkoxy of up to 10 carbon atoms, alkylthio of up to 10 carbon atoms, mono- and dialkylamino, halogen, —CN, —NO₂, alkylenedioxy, alkyl of up to 10 carbon atoms and aryl and its pharmaceutical salts sufficient to treat bone and cartilage disease and an inert pharmaceutical carrier. 